The objective of this project is to identify and characterize changes at the cellular and molecular levels responsible for the development of preneoplastic and neoplastic variants of rat tracheal epithelial (RTE) cells. To identify cells having critical preneoplastic changes, comparative transformation experiments are described in which independent populations of preneoplastic RTE cells are treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or with cloned oncogene-containing DNAs from Harvey murine sarcoma virus (HaMSV, ras), polyoma virus, and MC29 virus (myc). Differential responses of the various cell lines to these treatments reveal differences in their preneoplastic potential. Using the hypothesis that differential responses of "preneoplastic" cell lines to carcinogens or oncogenic DNAs have a genetic basis, efforts will be directed towards the molecular cloning of the genes responsible for the conversion of preneoplastic to neoplastic cells. These studies require the development of assays suitable for the detection of these genes. The assay, based on the use of preneoplastic RTE cells for identification of RTE cell tumor genes, provides an intraspecific system in which the genes will be identified in cells that are genetically related to the cells in which the gene was originally active. Transfections of normal RTE cells with cloned oncogene-containing DNAs (ras, polyoma) induced escape from senescence but induced no complete neoplastic transformation. The latter was induced by transfection of oncogenes in colonies previously altered by treatment with MNNG (in all cell lines with polyoma and in some lines only with ras), indicating a multistep process of transformation.